Methods And Compositions For Treatment Of Interferon-Resistant Tumors

ABSTRACT

The present invention provides a method for the treatment of interferon resistant bladder tumors through the use of a non-replicating agent which induces human cells to express interferon species. In particular it is noted that inducing interferon expression in the patient&#39;s body possesses properties not associated with recombinant produced, intravenously-administered interferon proteins. The present invention further provides compositions useful in the treatment of bladder cancer resistant to treatment with intravenous interferon polypeptide, by using a non-replicating agent which induces human cells to express interferon species, e.g., an antigenic, replication-deficient virus optionally carrying an interferon transgene.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.60/528,525, filed Dec. 10, 2003, which application is incorporatedherein by reference for all purposes.

BACKGROUND OF THE INVENTION

Interferon protein therapy is well established in the clinicalenvironment as a treatment for a variety of cancers. Two widely usedcommercially available interferon species produced by recombinant DNAtechnology are interferon α-2b recombinant (Intron A®, ScheringCorporation) and interferon α-2a recombinant (Roferon®, Hoffman LaRoche,Inc.). Intron A is indicated for use in the treatment of malignantmelanoma in combination with surgery, aggressive follicularNon-Hodgkin's Lymphoma in combination with anthracycline chemotherapy,intralesional treament of condylomata acuminata, hairy cell leukemia,and AIDS-Related Kaposi's Sarcoma. Roferon is indicated for use in thetreatment of Philadelphia chromosome positive chronic myelogenousleukemia (CML) and AIDS-related Kaposi's sarcoma.

Additionally, investigational approaches to the use of recombinantvectors encoding interferon species have been evaluated for anti-tumoreffects including the treatment of cancers of the ovary, kidney, andbladder, multiple myeloma, melanoma, certain lymphomas and leukemias,and Kaposi's sarcoma. The antitumor activities of replication deficientadenoviruses encoding interferon (Ad-IFNα) have been reported against avariety of human tumor xenografts following direct injection (Ahmed et.al., Cancer Gene Therapy, 8:788-95 (2001)), which included humantransitional bladder carcinoma (TCC) xenografts (Izawa et al., Clin.Cancer Res., 8:1258-1270 (2002)). These studies show that interferon canmediate both direct antitumor activity and a significant bystandereffect, characterized by the activation of host effector cells, enhancedapoptosis, and the inhibition of angiogenesis.

One particularly notable application where recombinant interferonprotein has been evaluated for efficacy is in the treatment of bladdercancer. Superficial bladder cancers are diagnosed in over 45,000patients/year in the United States. The disease is typicallycharacterized as a slowly progressing malignancy that originates fromthe surface lining of the urothelium. While most of these superficialcancers can be adequately managed with periodic transurethral resection(TUR) and surveillance, this is far from an optimal approach because 60to 70 percent of superficial tumors recur after TUR, and up to 30percent evolve into more aggressive, potentially lethal cancers. Thehigh recurrence rate and the unpredictability of the progressionpatterns have led to the widespread use of intravesical therapy forlocal control of the disease. Immunotherapy with intravesicallyinstilled Bacillus Calmette-Guérin (BCG) can usually delay diseaseprogression in newly diagnosed patients. Unfortunately this therapy doesnot produce a qualitative change in the underlying biology of the tumorand many patients remain at substantial risk of eventual progressing toinvasive, life-threatening cancer. In fact, even with close surveillanceand follow-up, at least 50% of patients will eventually recur, and 30%will die of metastatic bladder cancer, despite originally presentingwith “only” carcinoma in situ. Herr et al., J. Urol., 163:60-61 (2000)and Dalbagni et al., Urol. Clin. North Am., 27:137-146 (2000). Clearly,more effective intravesical therapies are necessary to improve overallsurvival and provide an alternative to radical cystectomy.

Intravesical interferon-α2b (IFNα2b) recombinant (Intron A) is welltolerated as a monotherapy in superficial bladder cancer patients and isan approved indication for this agent in many countries. Intron A hasdemonstrated dose-related clinical efficacy as a salvage therapy in thescenario of BCG failure, although the durability of the response islimited and most patients relapse within the first year of treatment.Belldegrun et al., J. Urol., 159(6):1793-801 (1998). Recently, Intron Ahas been combined with BCG in an attempt to enhance the cellular immuneresponse to BCG and improve the response to therapy. O'Donnell et al.,J. Urol., 166:1300-1304 (2001). Combination therapy has been effectivein BCG refractory TCC, but many of these initial responders relapse withsuperficial disease and ultimately require cystectomy.

Gene therapy employing recombinant vectors encoding interferon speciesprovides a promising alternate approach to the treatment of refractorysuperficial bladder cancer. Local delivery can maximize transgeneexpression in the urothelium and minimize vector distribution to vitalorgans outside of the bladder. In addition, efficient transfer of a geneencoding a secreted protein (e.g., IFNα2b) to both normal and malignantcells of the urothelium can generate a potent anti-tumor bystandereffect coincident with sustained local protein concentrations in theurine.

SUMMARY OF THE INVENTION

The present invention provides methods for the treatment of interferonresistant tumors through the use of recombinant vectors encodinginterferon species. In particular, it is noted that interferon speciesprovided by recombinant vectors possess properties not associated withthe available recombinantly produced interferon proteins. The presentinvention further provides compositions useful in the treatment ofinterferon resistant tumors using recombinant vectors encodinginterferons.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a data of the effect of intravestical Ad-IFN-α2α1/Syn 3or Ad-IFN-α2b/Syn3 on tumor regression. The graph at the left of thepanel shows marked decrease in the pre-treatment tumor size 21 daysafter Ad-IFNα2α1/Syn 3 or Ad-IFNα2b/Syn3 treatment*, P=0.0024. All othertreatment groups showed progressive tumor growth including Ad-IFNα2α1without Syn3 or the IFN-α2α1 protein alone. To the right are a series offluorescence micrographs providing a representative example ofpre-treatment tumor images and those taken 21 days later from individualmice in each treatment group.

FIG. 2 provides a demonstration of prolonged IFN expression followingAd-IFN/Syn3 intravesical treatment. Panel A demonstrates that sustainedlevels of IFN are seen in the bladder homogenates of athymic micetreated once with Ad-IFNα2b/Syn3 which was increased after twoexposures. In contrast, similar treatment with 200,000 IU of Intron Aprotein itself did not show any such high or prolonged IFN levels.Arrows indicate when treatment 1 and 2 were given. Panel B provides dataindicating that the same high levels of IFN in the urothelium andadjacent bladder tumor is seen by immunohistochemical analysis 2 daysafter two treatments with Ad-IFNα2α1/Syn 3 in athymic mice. Arrows showstrong perinuclear IFN staining present in many cells. Panel Cdemonstrates that similar high levels of IFN are seen in the urotheliumof outbred Balb-C mice 7 days after treatment with either Ad-IFNα2α1/Syn3 or Ad-IFNα2b/Syn3. No morphological changes in the urothelium wereseen by histological examination or H&E stained sections.

FIG. 3 provides a comparison of Ad-IFN cytotoxicity to bladder cancercell lines resistant to over 100,000 IU of Intron A. Panel A shows thenegative IFN staining in control cells (10× Magnification) and markedperinuclear IFN staining in 253J-BV cells treated with Ad-IFNα2α1 (40×Magnification). Panel B is data from a flow cytometric analysis showingincrease in the number of subdiploid cells at 48 hrs after treatment ofa 50 MOI Ad-IFNα2α1 in 253J B-V cells. Red bars indicate the subdiploidpopulation. Panel C illustrates an increase in the number of G2/M cellsfollowing Ad-IFNα2α1 treatment to over 60% was observed. Panel Ddemonstrates the marked cytotoxicity to Ad-IFNα2α seen in the 253J B-Vcells at the same 50 MOI of Ad-IFNα2α1. A 50 MOI of Ad-βgal was alsoused for these studies as a control and no cytotoxicity was seen, norwere changes observed in these cells when treated with 100,000 IU ofIntron A. Similar results were observed using KU7 cells or usingAd-IFN-α2b.

FIG. 4 illustrates caspase dependent cell death occurs in bladder cancercells resistant to IFN protein following Ad-IFN treatment includingcells which show no IFN staining. Panel A provides KU7 or 253J-BV cellstreated with Ad-IFN-α2α1. Two by two optical channels merged images oftriple staining of cells for IFN (red) and DNA (blue) upper panels andfor active caspase-3 (green) and DNA (blue) lower panels are shown 36 hafter treatment. Caspase activation and nuclear condensation is seen incells expressing IFN (white arrows). Caspase activation, however, isalso seen in IFN-nonexpressing cells found in close proximity ofIFN-expressing cells (yellow arrows), demonstrating a bystander effectof IFN expression in the two different cellular populations. Panel Bprovides quantitation of caspase-3 activation in BV and KU7 cells 72 hrafter Ad-IFN treatment at the same MOI.

FIG. 5 is a comparison of Ad-IFN and Intron A on bladder cancer cells.Panel A illustrates the high and sustained production of IFN in thesupernatant from B-V and KU7 following Ad-IFN treatment at two MOIs.Levels reached 10 million pg/ml (˜2.5 million IU of IFN) in both celllines. The addition of 400,0000 pg/ml (100,000 IU/ml) of Intron A to themedium of each cell line is shown for comparison and remained constantover a 6 day period. Panel B illustrates the percentage of subdiploidcells shown 48 and 72 hours after addition of 2.5 million IU/ml ofIntron A or 50 MOI of Ad-IFN-α2b. Panel C illustrates the lack ofmorphological changes or IFN staining in KU7 cells seen after Intron Atreatment at the same concentration and time points as Panel B. Incontrast, note the cells with increased size and strong perinuclearstaining following treatment with Ad-IFN-α2b along with several smallapoptotic appearing cells (arrows) (Magnification 40×).

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The present invention provides a method of treating aninterferon-resistant tumor by contacting the tumor with a recombinantvector encoding an interferon such that the cells of the tumortransduced by the vector express the interferon encoded by the vector.

As used herein, the term “interferon-resistant tumor” refers to a tumorthat is refractory to treatment by the administration of interferonprotein in accordance with conventional interferon treatment protocols.In the case of bladder cancer, a tumor is considered refractory tointerferon treatment if the tumor fails to diminish in a course oftherapy of intravesical administration of up to six weeklyadministrations of approximately 100 million units of Intron A for anexposure time of approximately 1 hour. In the case of malignantmelanoma, a tumor is considered to be refractory to interferon treatmentif the tumor fails to respond following a treatment regimen comprisingthe intravenous administration of approximately 20 MIU/m² of Intron Afor 5 consecutive days per week for 4 weeks (induction phase) followingby a maintenance phase of 3 times per week for up to 48 weeks at a doseof approximately 10 MIU/m². In the case of diffuse diseases such aslymphomas and leukemias, there is not generally a localized tumor mass,but rather a systemic disease characterized by neoplastic white bloodcells. For purposes of the present invention, neoplastically transformedcells shall be considered tumors. In the case of follicular lymphomas,the tumor is considered refractory to interferon treatment if theindividual fails to respond to a treatment regimen comprisingsubcutaneous administration of 5 MIU of Intron A three times per weekfor up to 18 months. In the case of Ph-positive chronic myologenousleukemia (CML), the tumor is considered refractory to treatment if theindividual fails to respond to a treatment regimen consisting of a dailyintramuscular or subcutaneous injection of approximately 9 MIU ofRoferon for a treatment period of up to 5 to but as long as 18 months.Alternative to clinical evaluation and response to conventionalinterferon protein therapy, tumors may be identified as being refractoryto administration of interferon protein as determined in vitro inaccordance with conventional in vitro assay procedures. Characterizationof a tumor as an interferon-resistant tumor may be determined byexposure of the interferon-resistant tumor cells comprising the tumorbeing resistant to growth inhibition by contact with at least 100,000units of Intron A protein as determined in substantial accordance withthe MTT assay described in Example 6 herein.

The term “tumor” refers to a collection of neoplastic cells. The term“neoplastic cell” refers to a cell displaying an aberrant growthphenotype characterized by independence of normal cellular growthcontrols. As neoplastic cells are not necessarily replicating at anygiven time point, the term neoplastic cells comprise cells which may beactively replicating or in a temporary nonreplicative resting state (G1or G0). Localized populations of neoplastic cells are referred to asneoplasms or tumors (these terms are considered interchangeable). Aspreviously noted, the term tumors as used herein may also refer todiffuse neoplastic diseases such as leukemia where no substantiallocalized tumor mass is present. Neoplasms or tumors may be malignant orbenign. Malignant tumors are also referred to as cancers. In preferredembodiments, the cancer is an epithelial tumor, e.g., breast, lung,prostate, colorectal, kidney, stomach, bladder or ovarian, or any cancerof the gastrointestinal tract.

While most tumors are generally monoclonal in origin, a tumororiginating from a monoclonal source may differentiate over time.Consequently, a tumor (including diffuse tumors) may possess individualcells or cell subpopulations which are interferon-resistant and cells orcell subpopulations that are sensitive to interferon treatment. The terminterferon-resistant tumors refers to tumors where a substantialpopulation (i.e., at least 20%) of the cells which comprise the tumorsare interferon-resistant as determined in accordance with the MTT assaydescribed in Example 6 herein.

The term “treatment,” as used herein, is intended to refer to theintroduction of nucleic acid encoding an interferon to a patient for thepurpose of exposing a tissue of interest comprising neoplastic cells tointerferon. Thus, for example, a “cancerous” tissue is intended to referto a tissue in which one or more cells is classified as cancerous,malignant, tumorous, precancerous, transformed, or as an adenoma orcarcinoma, or any other synonym commonly used in the art for theseconditions. A treatment may be considered therapeutic if the treatmentindicates an improvement in the physiological or psychological conditionof the subject in which the neoplasm is present. Improvement in thecondition may be measured by a variety of means including reduced tumorburden, reduction of tumor progression, slowing of tumor progression,increased survival, tumor regression, amelioration symptoms associatedwith tumor burden including but not limited to fatigue, anorexia, pain,depression, neutropenia and cognitive dysfunction.

As used herein, the term “interferon” (abbreviated “IFN”) referscollectively to type 1 and type 2 interferons including deletion,insertion, or substitution variants thereof, biologically activefragments, and allelic forms. As used herein, the term interferon(abbreviated “IFN”) refers collectively to type 1 and type 2interferons. Type 1 interferon includes interferons-α, -β and -ω andtheir subtypes. Human interferon-α has at least 14 identified subtypeswhile interferon-β has 3 identified subtypes. Particularly, preferredinterferon-alphas include human interferon alpha subtypes including, butnot limited to, α-1 (GenBank Accession Number NP 076918), α-1b (GenBankAccession Number AAL35223), α-2, α-2a (GenBank Accession NumberNP000596), α-2b (GenBank Accession Number AAP20099), α-4 (GenBankAccession Number NP066546), α-4b (GenBank Accession Number CAA26701),α-5 (GenBank Accession Numbers NP 002160 and CAA26702), α-6 (GenBankAccession Number CAA26704), α-7 (GenBank Accession Numbers NP 066401 andCAA 26706), α-8 (GenBank Accession Numbers NP002161 and CAA 26903), α-10(GenBank Accession Number NP 002162), α-13 (GenBank Accession Numbers NP008831 and CAA 53538), α-14 (GenBank Accession Numbers NP 002163 and CAA26705), α-16 (GenBank Accession Numbers NP 002164 and CAA 26703), α-17(GenBank Accession Number NP 067091), α-21 (GenBank Accession NumbersP01568 and NP002166), and consensus interferons as described inStabinsky, U.S. Pat. No. 5,541,293, issued Jul. 30, 1996, Stabinsky,U.S. Pat. No. 4, 897,471, issued Jan. 30, 1990, and Stabinsky, U.S. Pat.No. 4,695,629, issued Sep. 22, 1987, the teachings of which are hereinincorporated by reference, and hybrid interferons as described inGoeddel et al., U.S. Pat. No. 4,414,150, issued Nov. 8, 1983, theteachings of which are herein incorporated by reference. Type 2interferons are referred to as interferon γ (EP 77,670A and EP 146,354A)and subtypes. Human interferon gamma has at least 5 identified subtypes,including interferon omega 1 (GenBank Accession Number NP 002168).Construction of DNA sequences encoding inteferons for expression may beaccomplished by conventional recombinant DNA techniques based on thewell-known amino acid sequences referenced above and as described inGoeddel et al., U.S. Pat. No. 6,482,613, issued Nov. 19, 2002, theteachings of which are herein incorporated by reference.

“Biologically active” fragments of interferons may be identified ashaving any anti-tumor or anti-proliferative activity as measured bytechniques well known in the art (see, for example, Openakker et al.,supra; Mossman, J. Immunol. Methods, 65:55 (1983) and activate IFNresponsive genes through IFN receptor mediated mechanisms. Soluble IFN-αand IFN-β proteins are generally identified as associating with the Type1 IFN receptor complex (GenBank Accession Number NP 000865) and activatesimilar intracellular signaling pathways. IFN-γ is generally identifiedas associating with the type II IFN receptor. Ligand-induced associationof both types of IFN receptors results in the phosphorylation of thereceptors by Janus kinases subsequently activating STATs (signaltransducers and activators of transcription) proteins and additionalphosphorylation events that lead to the formation of IFN-inducibletranscription factors that bind to IFN response elements presented inIFN-inducible genes. Polypeptides identified as activating the IFNpathways following association with Type 1 and/or Type 2 IFN receptorsare considered interferons for purposes of the present invention.

The term “recombinant vector” refers to viral and nonviral vectorscomprising an interferon expression cassette that has been preparedusing conventional recombinant DNA technology. The term “interferonexpression cassette” is used herein to define a nucleotide sequencecontaining regulatory elements operably linked to the interferon codingsequence so as to result in the transcription and translation of aninterferon in a cell. The term “regulatory element” refers to promoters,enhancers, transcription terminators, polyadenylation sites, and thelike. The expression cassette may also contain other sequences aidingexpression and/or secretion of the interferon gene. The regulatoryelements may be arranged so as to allow, enhance or facilitateexpression of the interferon only in a particular cell type. Forexample, the expression cassette may be designed so that the interferonis under control of an inducible promoter, tissue specific or tumorspecific promoter, or temporal promoter. In general, the interferon isprovided in an expression vector comprising the following elementslinked sequentially at appropriate distances for functional expression:a promoter, an initiation site for transcription, a 3′ untranslatedregion, a 5′ mRNA leader sequence, a nucleic acid sequence encoding aninterferon polypeptide, and a polyadenylation signal.

The term “operably linked” refers to a linkage of polynucleotideelements in a functional relationship. A nucleic acid sequence is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. Operably linked means that the nucleotide sequencesbeing linked are typically contiguous. However, as enhancers generallyfunction when separated from the promoter by several kilobases andintronic seqences may be of variable lengths, some polynucleotideelements may be operably linked, but not directly flanked and may evenfunction in trans from a different allele or chromosome.

The term “inducible promoter” refers to promoters that facilitatetranscription of the interferon transgene preferably (or solely) undercertain conditions and/or in response to external chemical or otherstimuli. Examples of inducible promoters are known in the scientificliterature (see, e.g., Yoshida et al., Biochem. Biophys. Res. Comm.,230:426-430 (1997); Iida et al., J. Virol., 70(9):6054-6059 (1996);Hwang et al., J. Virol., 71(9):7128-7131 (1997); Lee et al., Mol. Cell.Biol., 17(9):5097-5105 (1997); and Dreher et al., J. Biol. Chem.,272(46):29364-29371 (1997). Examples of radiation inducible promotersinclude the EGR-1 promoter. Boothman et al., volume 138, supplementpages S68-S71 (1994).

Tissue specific and tumor specific promoters are well known in the artand include promoters active preferentially in smooth muscle (α-actinpromoter), pancreas specific (Palmiter et al., Cell, 50:435 (1987)),liver specific (Rovet et al., J. Biol. Chem., 267:20765 (1992); Lemaigneet al., J. Biol. Chem., 268:19896 (1993); Nitsch et al., Mol. Cell.Biol., 13:4494 (1993)), stomach specific (Kovarik et al., J. Biol.Chem., 268:9917 (1993)), pituitary specific (Rhodes et al., Genes Dev.,7:913 (1993)), prostate specific (Henderson et. al., U.S. Pat. No.5,698,443, issued Dec. 16, 1997), etc.

The term “temporal promoters” refers to promoters that drivetranscription of the interferon transgene at a point later in the viralcycle relative to the promoter controlling expression of the responseelement and are used in conjunction with viral vector systems. Examplesof such temporally regulated promoters include the adenovirus major latepromoter (MLP), and other late promoters. For herpes simplex viruses, anexample of temporal promoter includes the latent activated promoters.

The term “nonviral vector” refers to an autonomously replicating,extrachromosomal circular DNA molecules, distinct from the normal genomeand nonessential for cell survival under nonselective conditions capableof effecting the expression of an interferon coding sequence in thetarget cell. Plasmids autonomously replicate in bacteria to facilitatebacterial production, but it is not necessary that such plasmidscontaining the interferon coding sequence replicate in the target cellin order to achieve the therapeutic effect. Additional genes, such asthose encoding drug resistance, can be included to allow selection orscreening for the presence of the recombinant vector. Such additionalgenes can include, for example, genes encoding neomycin resistance,multi-drug resistance, thymidine kinase, β-galactosidase, dihydrofolatereductase (DHFR), and chloramphenicol acetyl transferase.

In order to facilitate delivery of the interferon gene to a particulartissue or organ, it may be advantageous to incorporate elements into thenonviral delivery system which facilitate cellular targeting. Forexample, a lipid encapsulated expression plasmid may incorporatemodified surface cell receptor ligands to facilitate targeting. Althougha simple liposome formulation may be administered, the liposomes eitherfilled or decorated with a desired composition of the invention candelivered systemically, or can be directed to a tissue of interest,where the liposomes then deliver the selected therapeutic/immunogenicpeptide compositions. Examples of such ligands include antibodies,monoclonal antibodies, humanized antibodies, single chain antibodies,chimeric antibodies or functional fragments (Fv, Fab, Fab′) thereof.Alternatively, nonviral vectors can be linked through a polylysinemoiety to a targeting moiety as described in Wu et al. U.S. Pat. No.5,166,320, issued Nov. 24, 1992, and Wu et al., U.S. Pat. No. 5,635,383,issued Jun. 3, 1997, the teachings of which are herein incorporated byreference.

The terms “viral vector” and “virus” are used interchangeably herein torefer to any of the obligate intracellular parasites having noprotein-synthesizing or energy-generating mechanism. The viral genomemay be RNA or DNA contained with a coated structure of protein of alipid membrane. The terms virus(es) and viral vector(s) are usedinterchangeably herein. The viruses useful in the practice of thepresent invention include recombinantly modified enveloped ornonenveloped DNA and RNA viruses, preferably selected frombaculoviridiae, parvoviridiae, picornoviridiae, herpesviridiae,poxviridae, or adenoviridiae. The viruses may be naturally occurringviruses or their viral genomes may be modified by recombinant DNAtechniques to include expression of exogenous transgenes and may beengineered to be replication deficient, conditionally replicating orreplication competent. Chimeric viral vectors which exploit advantageouselements of each of the parent vector properties (see, e.g., Feng, etal., Nature Biotechnology, 15:866-870 (1997)) may also be useful in thepractice of the present invention. Minimal vector systems in which theviral backbone contains only the sequences need for packaging of theviral vector and may optionally include a transgene expression cassettemay also be produced according to the practice of the present invention.Although it is generally favored to employ a virus from the species tobe treated, in some instances, it may be advantageous to use vectorsderived from different species that possess favorable pathogenicfeatures. For example, equine herpes virus vectors for human genetherapy are described in PCT International Publication No. WO98/27216,published Aug. 5, 1998. The vectors are described as useful for thetreatment of humans as the equine virus is not pathogenic to humans.Similarly, ovine adenoviral vectors may be used in human gene therapy asthey are claimed to avoid the antibodies against the human adenoviralvectors. Such vectors are described in PCT International Publication No.WO 97/06826, published Apr. 10, 1997.

The term “replication deficient” refers to vectors that are highlyattenuated for replication in a wild type mammalian cell. In order toproduce such vectors in quantity, a producer cell line is generallycreated by co-transfection with a helper virus or genomically modifiedto complement the missing functions. For example, HEK293 cells have beenengineered to complement adenoviral E1 deletions allowing propagation ofthe E1 deleted replication deficient adenoviral vectors in 293 cells.The term “replication competent viral vectors” refers to a viral vectorthat is capable of infection, DNA replication, packaging and lysis of aninfected cell. The term “conditionally replicating viral vectors” isused herein to refer to replication competent vectors that are designedto achieve selective expression in particular cell types. Suchconditional replication may be achieved by operably linking tissuespecific, tumor specific or cell type specific or other selectivelyinduced regulatory control sequences to early genes (e.g., the E1 geneof adenoviral vectors).

Cell type specificity or cell type targeting may also be achieved inviral vectors derived from viruses having characteristically broadinfectivities by the modification of the viral envelope proteins. Forexample, cell targeting has been achieved with adenovirus vectors byselective modification of the viral genome knob and fiber codingsequences to achieve expression of modified knob and fiber domainshaving specific interaction with unique cell surface receptors. Examplesof such modifications are described in Wickham et al., J. Virol.,71(11):8221-8229 (1997) (incorporation of RGD peptides into adenoviralfiber proteins); Amberg et al., Virology, 227:239-244 (1997)(modification of adenoviral fiber genes to achieve tropism to the eyeand genital tract); Harris et al., TIG, 12(10):400-405 (1996); Stevensonet al., J. Virol., 71(6):4782-4790 (1997); Michael et al., Gene Therapy,2:660-668 (1995) (incorporation of gastrin releasing peptide fragmentinto adenovirus fiber protein); and Ohno et al., Nature Biotechnology,15:763-767 (1997) (incorporation of Protein A-IgG binding domain intoSindbis virus). Other methods of cell specific targeting have beenachieved by the conjugation of antibodies or antibody fragments to theenvelope proteins (see, e.g., Michael et al., J. Biol. Chem.,268:6866-6869 (1993), Watkins et al., Gene Therapy, 4:1004-1012 (1997);Douglas et al., Nature Biotechnology, 14:1574-1578 (1996).Alternatively, particular moieties may be conjugated to the viralsurface to achieve targeting (see, e.g., Nilson et al., Gene Therapy,3:280-286 (1996) (conjugation of EGF to retroviral proteins). Targetingof vectors may also be achieved in accordance with the teaching ofMurphy, U.S. Pat. No. 6,635, 476, issued Oct. 21, 2003, the teachings ofwhich are herein incorporated by reference. These targetingmodifications may be introduced into the viral vectors of the presentinvention in addition to or in combination with other modifications tothe viral genome. Targeting modifications may be used with replicationdeficient, replication competent or conditionally replicating viruses.

Alternatively, cell type targeting with viral vectors may be achievedthrough the use of a pathway responsive promoter driving a repressor ofviral replication. The term “pathway-responsive promoter” refers to DNAsequences that bind a certain protein and cause nearby genes to respondtranscriptionally to the binding of the protein in normal cells. Suchpromoters may be generated by incorporating response elements which aresequences to which transcription factors bind. Such responses aregenerally inductive, though there are several cases where increasingprotein levels decrease transcription. Pathway-responsive promoters maybe naturally occurring or synthetic. Pathway-responsive promoters aretypically constructed in reference to the pathway of a functionalprotein which is targeted. For example, a naturally occurring p53pathway-responsive promoter would include transcriptional controlelements activated by the presence of functional p53 such as the p21 orbax promoter. Alternatively, synthetic promoters containing p53 bindingsites upstream of a minimal promoter (e.g., the SV40 TATA box region)may be employed to create a synthetic pathway-responsive promoter.Synthetic pathway-responsive promoters are generally constructed fromone or more copies of a sequence that matches a consensus binding motif.Such consensus DNA binding motifs can readily be determined. Suchconsensus sequences are generally arranged as a direct or head-to-tailrepeat separated by a few base pairs. Elements that include head-to-headrepeats are termed palindromes or inverted repeats and those withtail-to-tail repeats are termed everted repeats.

In the preferred practice of the invention, the viral vector is anadenovirus. The term “adenovirus” is synonomous with the term“adenoviral vector” and refers to viruses of the genus adenoviridiae.The term adenoviridiae refers collectively to animal adenoviruses of thegenus mastadenovirus including but no limited to human, bovine, ovine,equine, canine, porcine, murine and simian adenovirus subgenera. Inparticular, human adenoviruses includes the A-F sugenera as well as theindividual serotypes thereof the individual serotypes and A-F subgeneraincluding but not limited to human adenovirus types 1, 2, 3, 4, 4a, 5,6, 7, 8, 9, 10, 11 (Ad11A and Ad 11P), 12, 13, 14, 15, 16, 17, 18, 19,19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a,35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91. Theterm bovine adenoviruses includes, but is not limited to, bovineadenovirus types 1,2,3,4,7, and 10. The term canine adenovirusesincludes, but is not limited to, canine types 1 (strains CLL, Glaxo,RI261, Utrect, Toronto 26-61) and 2. The term equine adenovirusesincludes, but is not limited to, equine types 1 and 2. The term porcineadenoviruses includes, but is not limited to, porcine types 3 and 4. Theterm viral vector includes replication deficient, replication competent,and conditionally replicating viral vectors.

More preferred are vectors derived from human adenovirus types 2 and 5.These vectors may incorporate particular modifications to enhance theirtherapeutic potential. For example, they may include deletions of E1aand E1b genes. Certain other regions may be enhanced or deleted toprovide specific features. For example, upregulation of the E3 region isdescribed to reduce the immunogenicity associated with human adenoviralvectors administered to human subjects. The E4 region has beenimplicated as important to expression of transgenes from the CMVpromoter, however the E4orf 6 protein has been described as leading tothe degradation of p53 in target cells in the presence of E1b largeprotein. Steegenga et al., Oncogene, 16:345-347 (1998). Consequently,the elimination of such sequences of adenoviral vectors is preferred.

In one embodiment of the invention when using replication competentvectors to deliver the interferon gene, it is preferred that anadenoviral vector be employed containing specific deletions in the E1Aregion so as to reduce the ability of the E1a gene product to bind tothe p300 and Rb proteins while retaining the transactivating function ofthe E1a CR3 domain. The vectors of the present invention containdeletions in the E1a coding sequence to eliminate p300 and p105-Rbbinding sites in the 13S coding sequence. In one preferred embodiment ofthe invention, the p300 binding deletions are represented by deletionsof amino acids from about 4 to about 25 or from about 36 to about 49. Inone preferred embodiment of the invention, the Rb binding deletions arerepresented by elimination of amino acids from about 111-127, preferablyfrom about 111-123. More preferred is a vector wherein the deletion inthe E1a-p300 binding domain comprises a deletion of the codons for aminoacids 4 to 25 of the adenoviral E1a gene product. More preferred is avector wherein deletion in the E1a-Rb binding domain comprises adeletion of the codons for amino acids 111-123 of the adenoviral E1agene product. Alternatively, pRb binding may be eliminated by theintroduction of a mutation to eliminate amino acids 124-127 of the E1A289R and 243R proteins. In the most preferred embodiment of the presentinvention as exemplified herein the vector comprises a deletion of aminoacids 4-25 and 111-123 of the E1a 13S gene product.

Additionally, the elimination of amino acids from approximately 219 to289 of the E1a 289R protein and 173 to 243 of the E1A 243R protein maybe introduced. For example, by introducing a point mutation at aposition corresponding to position 1131 of the human Ad5 genome (i.e.,changing the cytosine¹¹³¹ to a guanine) creates a stop codon. Thismutation results in the elimination of amino acids 219 to 289 of the E1a289R protein and 173 to 243 of the E1A 243R protein. This mutation ismade optionally in addition to the deletions in Rb and p300 bindingdescribed above.

The practice of the present invention involves “contacting” the tumorwith a recombinant vector encoding interferon. Contact of the tumorcells is accomplished by exposure of the tumor cells with therecombinant vector such that the cells of the tumor are transduced bythe vector expressing the interferon encoded by the vector. Theparticular means by which the tumor is contacted by the vector willdiffer depending on the nature of the tumor to which the vector is beingdelivered. In some embodiments of the invention, the compositions of theinvention can be administered directly into a tissue by injection orinto a blood vessel supplying the tissue of interest. In furtherembodiments of the invention, where local administration is preferred,but where it is difficult to gain physical access to the tumor site, thecompositions are administered through catheters or other devices toallow access to a remote tissue of interest, such as an internal organ.The compositions of the invention can also be administered in depot typedevices, pumps, implants, or formulations to allow slow or sustainedrelease of the compositions. In further embodiments of the invention,the compositions of the invention are administered “locoregionally”,i.e., intravesically, intralesionally, and/or topically. In otherembodiments of the invention, particularly when treating diffuse tumors(such as leukemias) or metastatic disease, the compositions of theinvention may be administered systemically by intravenous orintraarterial routes of administration.

In order to demonstrate the performance of the present invention,recombinant vectors derived from human adenovirus type 5 were used toencode interferon species. Two adenovirus vectors containing IFNα geneswere evaluated in these studies. One vector contained the humaninterferon α2b gene (Ad-IFNα2b), and the other contained a chimerichuman “hybrid” interferon gene comprised of the human α2 N-terminusjoined to the human α1 C-terminus (Ad-IFNα2α1). Whereas IFN activity isgenerally species restricted, the hybrid interferon (aka ‘universal’ orIFN A/D) is active on cells derived from a variety of mammalian speciesincluding mouse and human. These two interferon vectors permittedevaluation of the effect of recombinant IFNα vectors on human tumors andthe broader effects of the hybrid interferon that can elicit responsesin both the cells of the murine and xenograft cells of the human tumorpresent in the mouse model.

To evaluate the response to intravestical IFN gene and protein therapy,an orthotopic bladder tumor model in nude mice was used. The model isdescribed in Watanabe et al., Cancer Gene Therapy, 7:1575-1580 (2000)and Zhou et al., Cancer Gene Therapy, 9:681-686 (2002). The amount oftumor found in histological sections of the bladders taken 21 days aftertreatment was highly correlative of the tumor burden calculated byimaging. Prior to intravesical treatment, the tumor burden of KU7 cellswithin the bladder of each mouse was imaged and quantitated two weeksafter the cancer cells were instilled in accordance with the teaching ofWatanabe et al. For intravesical instillation, the vectors wereadministered in conjunction with Syn3 (Example 1). The mice thenreceived various intravesical treatment for one hour on two consecutivedays as described in the Examples herein. Three weeks later the bladderswere imaged again for changes in tumor burden. As shown in FIG. 1 of theattached drawings, when either Ad-IFNα2α1/Syn3 or Ad-IFNα2b/Syn3 wasused, a marked tumor regression was observed (P<0.0024). In contrast,tumors grew rapidly following treatment with the control Ad-βgal/Syn3,Ad-IFNα2α1, or Syn3 alone. These results indicate that Syn3 markedlyenhanced the efficacy of adenoviral vectors encoding IFNα.

Because intravesical IFNα protein is used in clinical trials forsuperficial bladder cancer alone or in combination with BCG, experimentswere conducted to further delineate differences in activity of Ad-IFNversus the interferon protein therapy. The efficacy of the hybridinterferon protein was compared to Ad-IFNa2a1 in the orthotopic tumormodel described above. When 200,000 units (2 MIU/ml) of IFNα2α1 proteinwas given intravesically for 1 hour on 2 consecutive days, nosignificant tumor regression was noted. No antitumor effects weredetected either when IFNα2α1 protein was instilled on days 1, 7 and 14and evaluated for tumor burden 21 days after the first treatment. Theseresults are in contrast to the effect observed following Ad-IFNa2a1/Syn3therapy as demonstrated in FIG. 1. The urothelium appeared similar inall treatment groups including a mild degree of hyperplasia which wasrelated to the initial trypsinization of the urothelium required topromote initial tumor cell adhesion.

In order to evaluated the level and duration of interferon expressionfollowing intravesical AdIFNα/Syn3, a series of experiments wereconducted using nontumor-bearing athymic mice with the sameconcentrations of Ad-IFNα2b/Syn3 utilized for the foregoing efficacystudies, and compared a single intravesical treatment to two consecutivedays of exposure for one hour. The IFN protein in the bladder tissue wasassessed by harvesting the mouse bladders at various times aftertreatment and assaying for IFN protein present in tissue homogenatesusing an ELISA assay. One day after treatment, bladders containedapproximately 50,000 pg/mg of Intron A, which slowly decreased to5,000-10,000 pg/mg by 7d as shown in FIG. 2, Panel A of the attacheddrawings. In mice receiving a second Ad-IFNα2b/Syn3 treatment, tissueconcentrations reached 100,000 pg/mg and remained higher over a 7dperiod compared to the single exposure. In contrast, when 200,000 IU ofIFN protein was instilled, IFN concentrations measured in tissuehomogenates one hour after treatment were <1000 pg/mg, and no IFNprotein was detected at later times. High concentrations of IFN proteinwere found in the majority of urothelial cells by immunohistochemicalanalysis 2 days after either Ad-IFNα2α1/Syn3 or Ad-IFNα2b/Syn3 treatment(FIG. 2, Panel B). Similar staining was seen in many of the adjacenthuman tumor cells (FIG. 2, Panel B). In outbred Balb/C mice, strong IFNstaining could also be seen in many of the urothelial cells five daysafter treatment on two consecutive days with either Ad-IFNα2α1/Syn3 orAd-IFNα2b/Syn3 (FIG. 2, Panel C). In addition, the bladder appeared toshow little evidence of local toxicity upon histological examination(FIG. 2, Panel C). This is particularly important in the mice treatedwith Ad-IFNα2α1/Syn3 since the hybrid IFN is active in mouse cells.

In order to more fully illustrate the effect of vectors encodinginterferon in the treatment of interferon resistant tumors, a series ofexperiments were conducted to evaluate vectors encoding interferon ininterferon resistant tumor cell lines. Some human bladder cancer celllines are resistant to interferon induced cell death even after exposureto more than 100,000 IU/ml of either IFNα or IFNα proteins in vitro inaccordance with the MTT assay described herein. Certain cell interferonresistant tumor cell lines include the KU7 cells (which were used in theforegoing efficacy studies) and 253J B-V cells. When exposed torecombinant vectors encoding interferon, these interferon resistanttumor cell lines demonstrated sensitivity to Ad-IFNα treatment. Forexample, transduction of KU7 or 253J-BV cells with a 50 MOI ofAd-IFN-α2α1 or Ad-IFN-α2b resulted in elevated IFNα expression inapproximately 50% of cells as determined by immunocytochemical stainingfor IFN protein. A strong perinuclear IFN staining was often seen (FIG.3, Panel A) accompanied by morphological changes such as an increase insize and a marked decrease in the number of mitosis. This resulted insignificant cell cycle arrest at G₂/M (FIG. 3, Panels B and C), DNAfragmentation characteristic of apoptosis (FIG. 3, Panels B and C), andcytostasis (FIG. 3, Panel D).

To more fully elucidate the mechanism or mechanisms underlying theinduction of cell death of these interferon resistant tumor cell linesfollowing exposure to Ad-IFN, the effects of Ad-IFNα on caspase-3activation as an independent marker for apoptosis. Transduction withAd-IFNα stimulated marked activation of caspase-3 in both cell lines(FIG. 4, Panels A and B). Caspase-3 staining was seen in cellsexhibiting a high level of IFN-α expression as well as in many adjacentcells that showed no evidence of IFN-α expression (FIG. 4 a).Furthermore, when the percentage of caspase-3 positive cells wasquantitated in both 253J-BV and KU7 cell lines 72 hr after Ad-IFNtreatment at the same 50 MOI, over 70% of the cells were caspase-3positive (FIG. 4, Panel B). Exposure to Ad-IFNα resulted in substantialcytotoxicity, since no population of the cells regrew when cultured overextended periods. These results were similar in bladder cancer celllines evaluated, including all cell lines resistant to the IFNα protein.No such changes were seen in these cells following treatment with up to100,000 IU/ml of the IFNa protein or Ad-βgal at a similar MOI. Since nomore than 50% of the cells are transduced at this MOI, this alsoprovided additional evidence to support a strong Ad-IFN inducedbystander effect.

To examine the amount of IFN protein made in culture by Ad-IFN over timeboth KU7 and 253J B-V cells were treated with a 50 and 100 MOI ofAd-IFNα2b for 2.5 hr or with 100,000 IU/ml of Intron A. Whereas theconcentration of IFN protein in the supernatant remained constant atapproximately 400,000 pg/ml following Intron A (˜4 pg/IU) treatment,concentrations reached almost 10,000,000 pg/ml (˜2,500,000 IU) six daysafter treatment with 50 MOI of Ad-IFNα2b, and similar levels weredocumented by two days following exposure to 100 MOI of Ad-IFNα2b (FIG.5, Panel A).

Based on these results, the effects after the addition of 2,500,000IU/ml of Intron A were compared to that seen after treatment with a 50MOI of Ad-IFNα2b at 48 and 72 hr. An increase in the number ofsubdiploid cells was found in the Ad-IFN treated cells as had been notedpreviously, especially at 72 hr after infection, whereas no increase wasfound in the cells after Intron A exposure to such a high concentration(FIG. 5, Panel B). Similarly, no morphological changes or IFN stainingwere seen in cells 48 and 72 hr after the Intron A exposure compared tocontrol cells, whereas the typical morphological changes and IFNstaining were found in the Ad-IFN treated cells (FIG. 5, Panel C).Nevertheless, there was a significant increase in the percentage of G2/Mcells at 48 and 72 hr after Intron A treatment and this paralleled thechanges seen following Ad-IFN exposure. For example, in KU7 cells theG2/M population at 48 hr after treatment for the control, Intron A andAd-IFN groups were 9.7, 23.4 and 33.4, respectively, as well as 7.4,21.3 and 19.5 at 72 h.

The vectors encoding both the IFNα2b and hybrid interferons clearlydemonstrate that intravesical Ad-IFNα has potent antitumor activity.When tumor burden was compared before and after treatment, those tumorsthat received either Ad-IFNα2α1 or Ad-IFNα2b in a Syn3 formulationdisplayed a marked decrease in tumor size, whereas bladder tumors grewrapidly in the mice treated with Ad-β-gal/Syn3 as well as Ad-IFNα2α1,Syn3 or IFNα2α1 protein alone. Minimal local toxicity was also observedwith any treatment.

Although various bladder cancer cell lines, including the KU7 cells usedin the present in vivo experiments, were resistant to high levels of theIFNα protein itself in culture, they were uniformly killed in cellculture by Ad-IFNα in a caspase-dependent manner at particleconcentrations that resulted in transduction of only 50% or less ofcells. These results confirm that vectors encoding interferon speciessuch as Ad-IFNα possess strong bystander effects and the ability toovercome resistance to the IFN protein.

In summary, the foregoing experiments demonstrate that recombinantvector delivered interferons possess unique properties distinct fromcommercially available recombinant IFN protein and that vectors encodingIFN can overcome resistance to the IFN protein. With respect to bladdercancer, the studies clearly illustrate that intravesical instillation ofAd-IFN-α2α1/Syn3 or Ad-IFN-α2b/Syn3 for one hour on consecutive dayscaused a marked regression in the growth of human superficial bladdertumors in athymic mice when tumor burden was determined 3 weeks aftertreatment. Similar treatment with 200,000 IU per day of the IFN-α2α1protein did not result in any long-term IFN tissue levels and had noeffect on the growth of the tumors. Since Ad-IFN-α2α1/Syn3 andAd-IFN-α2b/Syn3 were comparable in producing cytotoxicity in vitro andtumor regression in vivo, the direct effect of sustained expression ofIFN was a substantial factor in tumor regression rather than its effecton the tumor microenvironment in this model because IFNα2b activatesonly interferon signaling pathways in human cells, although it can beproduced in both mouse and human cells. The data also demonstrates thatAd-IFNα can elicit caspase-dependent cytotoxicity in bladder cancercells resistant to high concentrations of the IFN protein. Apoptosis wasapparent not only in bladder cancer cells showing high intracellularlevels of IFNα by immunochemical staining but also in adjacent cellsthat did not demonstrate IFNα expression. These results demonstrate thattreatment with Ad-IFNa can produce a strong bystander effect inneighboring cells that may target tumor cells that escaped infection byAd-IFNα.

A potential concern for the clinical application of any agent is itspotential toxic side effects. However, these studies clearly demonstratea lack of toxicity associated with the administration of recombinantvectors encoding interferon, even though there is a high, persistentinterferon level produced by the vectors. The results from the foregoingstudies demonstrated no significant morphological changes in the normalurothelium when the athymic mice were examined three weeks afterAd-IFNα2α1/Syn3 treatment. In addition, when nontumor-bearing normaloutbred mice were similarly treated with Ad-IFNα2α1/Syn3 and examinedfor acute pathology in the bladder up to 21 days after exposure, nosignificant changes were identified upon histological examination of thebladder. In addition, pilot experiments in rats demonstrated thatalthough interferon transgene expression was high in the urine of rats,serum concentrations were minimal indicating low systemic exposure afterintravesical Ad-IFN treatment. These initial efficacy and toxicityresults suggest that Ad-IFNa2b/Syn3 will be well-tolerated.

The present invention further provides pharmaceutical formulationscomprising the vectors of the present invention. The compositions of theinvention will be formulated for administration by manners known in theart acceptable for administration to a mammalian subject, preferably ahuman. In particular, delivery systems may be formulated forintramuscular, intravenous, injectable depot type devices or topicaladministration.

In the case where nonviral gene delivery system is employed, theexpression plasmid containing the interferon gene may be encapsulated inliposomes. Liposomes include emulsions, foams, micelles, insolublemonolayers, liquid crystals, phospholipid dispersions, lamellar layersand the like. The delivery of DNA sequences to target cells usingliposome carriers is well known in the art. A variety of methods areavailable for preparing liposomes, as described in, e.g., Szoka et al.,Ann. Rev. Biophys. Bioeng., 9:467 (1980), Szoka, et al., U.S. Pat. No.4,394,448, issued Jul. 19, 1983, as well as U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369, incorporated herein by reference.Liposomes useful in the practice of the present invention may be formedfrom one or more standard vesicle-forming lipids, which generallyinclude neutral and negatively charged phospholipids and a sterol, suchas cholesterol. Examples of such vesicle forming lipids include DC-chol,DOGS, DOTMA, DOPE, DOSPA, DMRIE, DOPC, DOTAP, DORIE, DMRIE-HP,n-spermidine cholesterol carbamate and other cationic lipids asdisclosed in U.S. Pat. No. 5,650,096. The selection of lipids isgenerally guided by consideration of, e.g., liposome size, acid labilityand stability of the liposomes in the blood stream. Additionalcomponents may be added to the liposome formulation to increase serumhalf-life such as polyethylene glycol coating (so called “PEG-ylation”)as described in U.S. Pat. Nos. 5,013,556, issued May 7, 1991, and5,213,804, issued May 25, 1993.

The compositions of the invention will be formulated for administrationby manners known in the art acceptable for administration to a mammaliansubject, preferably a human. In some embodiments of the invention, thecompositions of the invention can be administered directly into a tissueby injection or into a blood vessel supplying the tissue of interest. Infurther embodiments of the invention the compositions of the inventionare administered “locoregionally,” i.e., intravesically,intralesionally, and/or topically. In other embodiments of theinvention, the compositions of the invention are administeredsystemically by injection, inhalation, suppository, transdermaldelivery, etc. In further embodiments of the invention, the compositionsare administered through catheters or other devices to allow access to aremote tissue of interest, such as an internal organ.

The compositions of the invention can also be administered in topicalformulations or polymer matrices, hydrogel matrices, polymer implants,or encapsulated formulations to allow slow or sustained release of thecompositions. When the delivery system is formulated as a solution orsuspension, the delivery system is in an acceptable carrier, preferablyan aqueous carrier. A variety of aqueous carriers may be used, e.g.,water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid andthe like. These compositions may be sterilized by conventional, wellknown sterilization techniques, or may be sterile filtered. Theresulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents and the like, for example, sodium acetate, sodium lactate, sodiumchloride, potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, etc.

The concentration of the compositions of the invention in thepharmaceutical formulations can vary widely, i.e., from less than about0.1%, usually at or at least about 2% to as much as 20% to 50% or moreby weight, and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.

In some applications, it is desirable to administer the recombinantvector in conjunction with enhancing agents that facilitate the transferof the nucleic acid encoding interferon to the target cell. Examples ofsuch delivery enhancing agents include detergents, alcohols, glycols,surfactants, bile salts, heparin antagonists, cyclooxygenase inhibitors,hypertonic salt solutions, and acetates. Alcohols include for examplethe aliphatic alcohols such as ethanol, N-propanol, isopropanol, butylalcohol, acetyl alcohol. Glycols include glycerine, propyleneglycol,polyethyleneglycol and other low molecular weight glycols such asglycerol and thioglycerol. Acetates such as acetic acid, gluconic acid,and sodium acetate are further examples of delivery-enhancing agents.Hypertonic salt solutions like 1M NaCl are also examples ofdelivery-enhancing agents. Bile salts such as taurocholate, sodiumtauro-deoxycholate, deoxycholate, chenodesoxycholate, glycocholic acid,glycochenodeoxycholic acid and other astringents such as silver nitratemay be used. Heparin-antagonists like quaternary amines such asprotamine sulfate may also be used. Anionic, cationic, zwitterionic, andnonionic detergents may also be employed to enhance gene transfer.Exemplary detergents include but are not limited to taurocholate,deoxycholate, taurodeoxycholate, cetylpyridium, benalkonium chloride,Zwittergent 3-14 detergent, CHAPS(3-[(3-Cholamidopropyl)dimethylammoniol]-1-propanesulfonate hydrate),Big CHAP, Deoxy Big CHAP, Triton-X-100 detergent, C12E8,Octyl-B-D-Glucopyranoside, PLURONIC-F68 detergent, Tween 20 detergent,and TWEEN 80 detergent (CalBiochem Biochemicals). Particularly preferredenhancing agents and methods are described in Engler et al., U.S. Pat.No. 6,312,681, issued Nov. 6, 2001, Engler et al., U.S. Pat. No.6,165,779, issued Dec. 26, 2000, and Engler et al., U.S. Pat. No.6,392,069, issued May 21, 2002, the entire teachings of which are hereinincorporated by reference. A particularly preferred enhancing agentuseful in the practice of the present invention is a compound termedSyn3 of the Formula I:

Additional enhancing agents useful in the practice of the presentinvention include, but are not limited to, the compounds of the FormulasII, III, IV, and V and their pharmaceutically acceptable salts:

Initial efforts to transduce the urothelium using adenovirus vectors hadlimited success, due in part to the presence of an anti-adherencebarrier that protects against infections. Pagliaro et al., J. Clin.Oncol., 15:2247-2253 (2003). An excipient, Syn3, has been identifiedthat dramatically enhances adenovirus transduction of the urothelium.Connor et al., Gene Ther., 8:41-8 (2001); Yamashita et al., Cancer GeneTherapy, 9:687-691 (2002). In animal models, intravesical administrationof adenovirus vectors in a Syn3 formulation markedly increased transgeneexpression in both normal urothelium and superficial bladder tumors. Theenhancing effects of Syn3 persist over a time window of approximately 1hour. Consequently, the recombinant vectors of the present inventionwhen used in conjunction with Syn3 are preferably administeredcontemporaneously and generally within a period of approximately onehour following exposure of the bladder to Syn3.

The present invention further provides a method of treating a mammalianorganism containing a tumor by administering to the mammalian organism apharmaceutical formulation comprising a recombinant vector encoding aninterferon. The term “mammalian organism” includes, but is not limitedto, humans, pigs, horses, cattle, dogs, and cats. When the vector is arecombinant adenoviral vector, preferably one employs an adenoviralvector endogenous to the mammalian type being treated. Although it isgenerally favored to employ a virus from the species to be treated, insome instances, it may be advantageous to use vectors derived fromdifferent species which possess favorable pathogenic features. Forexample, it is reported (PCT International Publication No. WO 97/06826,published Apr. 10, 1997) that bovine adenoviral vectors may be used inhuman gene therapy to minimize the immune response characteristic ofhuman adenoviral vectors. By minimizing the initial immune response,rapid systemic clearance of the vector is avoided resulting in a greaterduration of action of the vector. A pre-exisiting or inducedimmunological response to repeated in vivo administration of viralvectors has been associated with a diminished therapeutic effect ofsystemically administered viral vectors through multiple courses ofadministration. While this can be overcome by adjustments to dosage, onemay also administered the vectors of the present invention incombination with immunosuppressive agents. Examples of immunosuppressiveagents include, but are not limited to, cyclosporine, azathioprine,methotrexate, cyclophosphamide, lymphocyte immune globulin, antibodiesagainst the CD3 complex, adrenocorticosteroids, sulfasalzaine, FK-506,methoxsalen, and thalidomide. Alternatively, transient elimination ofantiviral antibodies may be accomplished in accordance with the teachingof La Face et al., U.S. Pat. No. 6,464,976, issued Oct. 15, 2002, theteaching of which is herein incorporated by reference.

The determination of the optimal dosage regimen for treatment of thedisease will be based on a variety of factors which are within thediscretion of the attending health care provider, such as theprogression of the disease at the time of treatment, age, weight, sex,the type of vector being employed, whether it is being formulated with adelivery enhancing agent, the frequency of administration, etc. However,recombinant adenoviral vectors have been demonstrated to be safe andeffective in human beings in the dosage range between 1×10⁵ and 1×10¹²viral particles per dose in a multiple dosing regimen over a period ofseveral weeks. Consequently, administration of recombinant adenoviralvectors encoding interferon may be used in such dosage ranges.

In the preferred practice of the present invention for the treatment ofsuperficial bladder cancer in human beings, course of treatmentcomprising a dose of from 1×10¹⁰/ml to 1×10¹²/ml (most preferablyapproximately 1×10¹¹/ml) recombinant adenoviral particles encodinginterferon α2b in a volume of approximately 100 ml is instilledintravesically for a period of approximately one hour. An alternatecourse of treatment comprises a dose of from 1×10¹⁰/ml to 1×10¹²/ml(most preferably approximately 1×10¹¹/ml) recombinant adenoviralparticles encoding interferon α2b in a volume of approximately 100 ml isinstilled intravesically for a period of approximately one hour followedby a second substantially equivalent dose within 7 days, 5 days, 4 days,3 days, 2 days or on consecutive days following the first dose. Eachcourse of treatment is repeatable, depending on the course of diseaseprogression. In the case of intravesically administered recombinantvectors for the treatment of bladder cancer, optimal interferon geneexpression is generally observed when the courses of treatment aredistanced by at least 14 days, more preferably about 30 days, and mostpreferably about 90 days.

In the preferred practice of the invention for the treatment ofhepatocellular carcinoma in a human being, a dosage regimen comprisingapproximately 1×10¹⁰-1×10¹² particles of a replication deficientrecombinant adenoviral vector expressing an intracellular interferonspecies is administered intratumorally or via the hepatic artery for aperiod of five to seven consecutive days. This dosage regimen may berepeated over a course of therapy of approximately three to six weeks. Aparticularly preferred dosage regimen for the treatment ofhepatocellular carcinoma in a human subject suffering therefrom would beto provide intrahepatic arterial administration of from approximately1×10¹⁰-1×10¹² particles of a replication deficient recombinantadenoviral vector expressing inteferon-α2b under control of the AFPpromoter for approximately five consecutive days. Most preferably, thisdosage regimen is carried out in parallel with other chemotherapeuticregimens.

In the situation where the vector is a replication competent vector, thedosage regimen may be reduced. For example, a replication competentadenoviral vector may be constructed wherein the replication issubstantially restricted to hepatocellular carcinoma cells by using theAFP promoter (for example) to drive expression of E1 proteins in lieu ofthe native E1 promoter. Such vector would preferentially replicate inand express interferon in tumor cells and possess the desirable abilityto spread to surrounding cells expanding the therapeutic effect andallowing for a reduced dosage or shorter duration of treatment.

The compositions and methods of the present invention may be practicedalone or in combination with conventional chemotherapeutic agents ortreatment regimens. Examples of such chemotherapeutic agents includeinhibitors of purine synthesis (e.g., pentostatin, 6-mercaptopurine,6-thioguanine, methotrexate) or pyrimidine synthesis (e.g., Pala,azarbine), the conversion of ribonucleotides to deoxyribonucleotides(e.g., hydroxyurea), inhibitors of dTMP synthesis (5-fluorouracil), DNAdamaging agents (e.g., radiation, bleomycines, etoposide, teniposide,dactinomycine, daunorubicin, doxorubicin, mitoxantrone, alkylatingagents, mitomycin, cisplatin, procarbazine) as well as inhibitors ofmicrotubule function (e.g., vinca alkaloids and colchicine).Chemotherapeutic treatment regimens refers primarily to nonchemicalprocedures designed to ablate neoplastic cells such as radiationtherapy. These chemotherapeutic agents may be administered seperately ormay be included with the formulations of the present invention forco-administration. The present invention may also be practiced incombination with conventional immunotherapeutic treatment regiments suchas BCG in the case of superficial bladder cancer.

EXAMPLES

The following examples are considered illustrative of the practice ofthe present invention and should not be considered as limiting of thescope thereof.

Example 1 Cell Lines, Vectors, and Reagents

The bladder cancer cell lines, KU7/GFP clone 6 and 253J-B-V, were usedfor these studies. KU7/GFP clone 6 is stably transfected with thegreen-flourescent protein and was used for all in vivo studies. Thesecell lines are described in Watanabe et al., Cancer Gene Therapy,7:1575-1580 (2000). The cells were grown in modified minimum essentialmedium supplemented with 10% FCS and incubated at 37° C. in 5% CO₂ and95% air.

Recombinant adenoviral vectors encoding interferon α2α1 were prepared insubstantial accordance with the teaching of Ahmed et al., InterferonCytokine Res., 21(6):399-408 (2001). Recombinant adenoviral vectorsencoding IFN-α2b and β-galactosidase (β-gal were prepared in substantialaccordance with the teaching of Ahmed et al., Hum. Gene Ther.,10(1):77-8 (1999) and are also described in Gregory et al., U.S. Pat.No. 6,210,939, issued Apr. 3, 2001, the entire teaching of which isherein incorporated by reference.

Intron A protein (interferon α-2b recombinant) is commercially availablefrom Schering Corporation.

Syn3 was prepared in substantial accordance with the teaching of Engleret al., U.S. Pat. No.6,392,069, incorporated, supra.

Example 2 Superficial Tumor Formation, Treatment and Imaging

The methods for growing superficial KU7-GFP human bladder tumors inathymic mice and their imaging were conducted as previously beendescribed in Izawa et al., Clin. Cancer Res, 8:1258-1270 (2002) and Zhouet al., Cancer Gene Therapy, 9:681-686 (2002). Briefly, two weeks afterthe tumor cells were instilled, the bladders were imaged for thepresence of GFP—containing tumor. Mice then received an intravesicalinstillation of 100 μl of either Ad-IFN-α2α1/Syn3, Ad-IFN-α2b/Syn3,Ad-IFN-α2α1, Ad-β-gal/Syn3, Syn3 or the IFN-α2α1 protein for 1 hour. Apurse-string suture was tied around the urethra to ensure retentionduring the procedure. Virus, Syn3 and interferon protein concentrationswere 1×10¹¹ P/ml, 1 mg/ml and 2×10⁵ IU/100 μl, respectively. Between 6to 8 mice were treated per group except for Ad-IFN-α2α1/Syn3 andAd-IFN-α2α1 groups in which 14 and 16 mice were treated, respectively.

Three weeks after the first treatment the bladder was reexposed andimaged. Following euthanasia each bladder was subsequentlyintravesically instilled with 100 μl of 10% formalin and the urethrasutured. The bladders were then removed, fixed in 10% formalin andembedded in paraffin blocks. Sections were then taken throughout thebladder for H & E staining to document the presence or absence of tumor,as well as to correlate the tumor imaging with histological evidence oftumor.

Example 3 Homogenate Gene Expression Studies

Female athymic mice were anesthetized, catheterized and received eithera single 100 μl intravesical administration of Intron A (2×10⁶ IU/ml) or1-2 daily treatments with Ad-IFN/Syn3 (1×10¹¹ P/m1:1 mg/ml) for 1 houras described above. At various intervals the bladders were harvested andfrozen. The bladders were later thawed and transferred into lysis buffer(Promega). Samples were homogenized for 20 sec (Fast Prep, Q-BIOgene).The homogenates were then assayed for human interferon a proteinconcentration using an ELISA (Endogen). Interferon protein concentrationwas expressed as pg IFN/mg bladder tissue.

Example 4 Immunochemical Analysis

For in vivo studies interferon immunohistochemistry was performed insubstantial accordance with the teaching of Izawa et al. above. Apositive reaction was indicated by a brown staining. Similarly,interferon staining for cells in culture was performed in substantialaccordance with the teaching of Xu et al., Oncogene, 4:807-812 (1989),except that the primary antibody used was a 1:500 dilution of rabbitpolyclonal antibody against human interferon α (Hu-IFN-α, PBL).

Example 5 Statistical Analysis

Statistical analysis was performed with Image-Pro® Plus version 4.0software for Windows (commercially available from Media Cybernetics,Inc., 8484 Georgia Avenue, Suite 200, Silver Spring, Md. 20910-5611 USA)to calculate the pixel of pre-treatment bladder area, pre-treatment GFParea, post-treatment bladder area and post-treatment GFP area.Subsequently, the percentages of pre-treatment GFP area overpre-treatment bladder area and post-treatment GFP area overpost-treatment bladder area were calculated. We performed anonparametric test (Kruskal-Wallis test) to assess differences in thepercent change in tumor size among the 7 treatments as described inKruskal and Wallis, Journal of the American Statistical Association,47:583-621, Conover W J, Practical Nonparametric Statistics. 3rd ed.,John Wiley & Sons, Inc., New York (1952). We used Monte-Carlo simulationto determine the p-values for the test. To adjust for the fact that wewere performing many tests to compare each treatment to the others, weused a Bonferroni correction to examine the significance level for eachof these tests (0.05/21=0.0024). For the MTT results, the analysis wasdone using the General Lineal Models of the Statistica software(commercially available from StatSoft, Inc., 2300 East 14th Street,Tulsa, Okla. 74104).

Example 6 MTT, Flow Cytometry, Caspase Activity, and Measurement of IFNin Supernatants

MTT assays were performed in substantial accordance with the teaching ofZhang et al., Cancer Res., 63:760-765 (2003). Briefly, the bladder tumorcells were infected with a given adenovirus at a 50 and 100 MOI for 2.5h. At different time points the medium was removed, and 200 μl of mediumwere added containing 1 mg/ml MTT. After 3 hours, the reaction wasstopped with 200 μl of N,N-dimethylformamide lysis buffer, and theresultant solution read at A⁵⁹⁵ with a microplate reader. Additional 60mm dishes were treated similarly or with Intron A and harvested for flowcytometry and caspase activity. Supernatants from a duplicate disheswere obtained daily to measure the concentration of IFN for cellsexposed to Ad-IFN-α2b or IntronA. Cell cycle analysis, percentage ofsubdiploid cells and caspase activity were determined in substantialaccordance with the teaching of Williams et al., Mol. Cancer Ther.,2:835-843 (2003). The Intron A concentration was measured using an ELISAin accordance with the teaching of Fujisawa et al, J. InterferonCytokine Research, 16:555-559 (19996) using an Endogen Brand Human IFNaELISA kit commercially available as Catalog No. EHIFNA from PierceBiotechnology, Inc., P.O. Box 117, 3747 N. Meridian Road, Rockford, Ill.61105 in substantial accordance with the manufacturer's instructions.

Example 7 Confocal Microscopy Analysis for Active Caspase and IFN□Localization and their Correlation with Nuclear Morphological Changes

Human bladder cell lines were cultured on coverslips in DMEMsupplemented with 10% fetal calf serum then infected with Ad-α-gal,IFN-α2α1 or Ad-IFN-α 2b at a 50 and 100 MOI. Thirty-six hourspost-infection, cells were fixed in ice-cold methanol then blocked in10% goat serum in PBS and incubated overnight with anti-active caspase-3monoclonal rabbit (commercially available as Catalog NumberRDI-CASP3ACTabRm from Research Diagnostics Inc., Pleasant Hill Road,Flanders N.J. 07836) and anti-IFN-α monoclonal mouse (commerciallyavailable as Catalog No. 31101-1 from PBL Biomedical Laboratories, 131Ethel Road West, Suite 6, Piscataway, N.J. 08854) antibodies atdilutions of 1:1,000 and 1:500, respectively. Alexa-488 labeled goatanti rabbit and Alexa-546 labeled goat anti-mouse antibodies(commercially available as Catalog Nos. 11101 and 11010, respectively,from Molecular Probes, 29851 Willow Creek Road, Eugene, Oreg. 97402)were then added together with the DNA dye, 633-Topro-3 (commerciallyavailable from Molecular Probes) for another hour. Mounted slides wereimaged using a confocal microscope (Zeiss model LSM-510) and pixels wereanalyzed in each channel using Argon and He/Ne lasers equipped withappropriate optical filters. The images are representative for at leastten microscopic fields analyzed for each condition. Each experiment wasperformed in duplicate and repeated at least three times.

1. A composition of matter comprising: a non-interferon agent whichinduces interferon production in human cells which are exposed to it,said agent being replication-deficient, said agent formulated in normalsaline as a non-emulsified suspension suitable for intravesicalirrigation, said formulation providing said agent in an amountsufficient to induce interferon production in human bladder cells, saidformulation further providing said agent in an amount effective to treatnon-muscle invasive bladder cancer.
 2. The composition of matter ofclaim 1, wherein said replication-deficient agent comprises at least onecomponent selected from the group consisting of replication-deficientvirus and antigen.
 3. The composition of claim 2 wherein saidreplication-deficient agent comprises antigen, said antigen comprisingviral antigen of a replication-deficient virus.
 4. The composition ofclaim 2 wherein said replication-deficient agent comprises anexpressible interferon transgene.
 5. The composition of claim 1, furthercomprising Syn3.
 6. The composition of matter of claim 1, wherein saidagent is provided in an amount sufficient to reduce the risk ofnon-muscle invasive bladder cancer resistant to, or recurrent after,intravesical treatment with live Bacillus Calmette-Guérin vaccine. 7.The composition of claim wherein said replication-deficient agentcomprises antigen.
 8. The composition of claim 7 wherein saidreplication-deficient agent is provided in an amount sufficient toreduce the risk of non-muscle invasive bladder cancer resistant to, orrecurrent after, intravesical treatment with live BacillusCalmette-Guérin vaccine.
 9. The composition of claim 5, wherein saidSyn3 and said agent are packaged together in a kit.
 10. A method oftreating a human with cancer which is resistant to, or recurrent after,treatment with intravenous interferon polypeptide, comprising:transducing cells of said with a vector comprising thereplication-deficient agent of claim 4 comprising an expressibletransgene coding for interferon polypeptide, in an amount sufficient forsaid transduced cells to express interferon in an amount sufficient totreat said cancer.
 11. The method of claim 10, wherein said vectorcomprises a recombinant virus.
 12. The method of claim 11, furthercomprising administering said virus with Syn3.
 13. A method of treatinga human with bladder cancer which is resistant to or recurrent afterintravesical treatment with live Bacillus Calmette-Guérin vaccine,comprising: intravesically administering the agent of claim
 1. 14. Amethod for delaying the progression of non-muscle invasive bladdercancer to muscular-invasive bladder cancer in a human patient,comprising: intravesically administering the agent of claim 1.